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The mitochondrial F1Fo ATP synthase complex has a key role in cellular energy metabolism. The general architecture of the enzyme is conserved among species and consists of a globular catalytic moiety F-1, protruding out of the inner side of the membrane, a membrane integral proton translocating moiety F-o, and a stalk connecting F-1 to F-o. The X-r...
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... F 1 F o ATP synthase, as illustrated in (Fig. 1), is an oligomeric complex whose general structure and *Address correspondence to this author at the Department of Medical Biochemistry and Biology-University of Bari, Piazza G. Cesare -Policlinico, 70124 Bari -Italy; Tel: +39-80-5478428; Fax: +39-80-5478429; E-mail: a.gaballo@biochem.uniba.it function are remarkably conserved among ...
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... inner membrane inside-out vesicles contributed to the knowledge of the topological organization of the stalk subunits. These data indicated that F o I-PVP(b), d, F6 and OSCP subunits are exposed at the matrix, but not at cytosolic side of the inner mitochondrial membrane, while subunits c and a are shielded to their antibodies on both sides [33] (Fig. 1). In E. coli, the a and c subunits are in contact and protons are thought to be translocated at the interface between them. Both subunits are highly conserved in all F ATPases [34,35]. Subunit A6L has its N-terminal part anchored to the membrane while the C-terminus is exposed at the matrix side; subunits f and g have both the ...
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... between them. Both subunits are highly conserved in all F ATPases [34,35]. Subunit A6L has its N-terminal part anchored to the membrane while the C-terminus is exposed at the matrix side; subunits f and g have both the N-terminus at the matrix side of the membrane, while the e subunit appears to be exposed essentially at the cytosolic side [36] ( Fig. 1). Subunit e has been shown to exist as a dimer in yeast [37] and rat liver mitochondrial ATP synthase [38]. It has also been suggested that this subunit plays a central role in the dimerization process, proposed for yeast [37] and mammalian [39] ATP synthase, as well as in the Ca 2+ dependent regulation of H + ATP synthase activity ...
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Citations
... However, some of them still have potential to be used as anticancer agents. For example, oligomycin A inhibits the proton-translocating Fo-portion of the ATP synthase, and also affects the F1-portion at high concentration [44,45]. Oligomycin dramatically attenuates BC metastatic seeding in the lungs, which demonstrates the functional importance of OXPHOS in metastasis and highlights its potential as a therapeutic target to prevent metastatic spread in patients with BC [46]. ...
Inhibitors of mitochondrial respiration and ATP synthesis may promote the selective killing of respiration-competent cancer cells that are critical for tumor progression. We previously reported that CADD522, a small molecule inhibitor of the RUNX2 transcription factor, has potential for breast cancer treatment. In the current study, we show that CADD522 inhibits mitochondrial oxidative phosphorylation by decreasing the mitochondrial oxygen consumption rate (OCR) and ATP production in human breast cancer cells in a RUNX2-independent manner. The enzyme activity of mitochondrial ATP synthase was inhibited by CADD522 treatment. Importantly, results from cellular thermal shift assays that detect drug-induced protein stabilization revealed that CADD522 interacts with both α and β subunits of the F1-ATP synthase complex. Differential scanning fluorimetry also demonstrated interaction of α subunits of the F1-ATP synthase to CADD522. These results suggest that CADD522 might target the enzymatic F1 subunits in the ATP synthase complex. CADD522 increased the levels of intracellular reactive oxygen species (ROS), which was prevented by MitoQ, a mitochondria-targeted antioxidant, suggesting that cancer cells exposed to CADD522 may elevate ROS from mitochondria. CADD522-increased mitochondrial ROS levels were enhanced by exogenously added pro-oxidants such as hydrogen peroxide or tert-butyl hydroperoxide. Conversely, CADD522-mediated cell growth inhibition was blocked by N-acetyl-l-cysteine, a general ROS scavenger. Therefore, CADD522 may exert its antitumor activity by increasing mitochondrial driven cellular ROS levels. Collectively, our data suggest in vitro proof-of-concept that supports inhibition of mitochondrial ATP synthase and ROS generation as contributors to the effectiveness of CADD522 in suppression of tumor growth.
... In shrimp cells, ATPsyn-β was shown to be located and activated on the cell surface of hematocytes and the cuticular epithelium cell membrane of gills (Liang et al., 2015), which satisfied the basic requirement to be a receptor for WSSV entrance into the host and may contribute to the systemic infection of the virus. Moreover, ATPsyn-β is a conserved protein in multiple species (Gaballo et al., 2002). The use of ATPsyn-β as a WSSV receptor provides a molecular explanation for the unusual broad host range of this virus. ...
White spot syndrome virus (WSSV) is highly virulent toward shrimp, and F1 ATP synthase β subunit (ATPsyn-β) has been suggested to be involved in WSSV infection. Therefore, in this study, interactions between Penaeus monodon ATPsyn-β (PmATPsyn-β) and WSSV structural proteins were characterized. Based on the results of yeast two-hybrid, co-immunoprecipitation, and protein pull-down assays, WSSV VP51B and VP150 were identified as being able to interact with PmATPsyn-β. Membrane topology assay results indicated that VP51B and VP150 are envelope proteins with large portions exposed outside the WSSV virion. Cellular localization assay results demonstrated that VP51B and VP150 co-localize with PmATPsyn-β on the membranes of transfected cells. Enzyme-linked immunosorbent assay (ELISA) and competitive ELISA results demonstrated that VP51B and VP150 bound to PmATPsyn-β in a dose-dependent manner, which could be competitively inhibited by the addition of WSSV virions. In vivo neutralization assay results further showed that both recombinant VP51B and VP150 could delay mortality in shrimp challenged with WSSV.
... The role of protons is also essential in prevention of futile ATP hydrolysis (Gaballo et al., 2002). In catalysis of ATP synthesis, the futile ATP hydrolysis by the F 1 F o complex is inhibited by the ATPase inhibitor protein (IF 1 ), which reversibly binds at one side of the F 1 F o connection. ...
The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i) the supply of ADP and Mg²⁺, supported by adenylate kinase (AK) equilibrium in the intermembrane space, (ii) the supply of phosphate via membrane transporter in symport with H⁺, and (iii) the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg²⁺ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg²⁺, adenylate transport, and phosphate release and supply.
... The role of protons is also essential in prevention of futile ATP hydrolysis (Gaballo et al., 2002). In catalysis of ATP synthesis, the futile ATP hydrolysis by the F 1 F o complex is inhibited by the ATPase inhibitor protein (IF 1 ), which reversibly binds at one side of the F 1 F o connection. ...
The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i) the supply of ADP and Mg2+, supported by adenylate kinase (AK) equilibrium in the intermembrane space, (ii) the supply of phosphate via membrane transporter in symport with H+, and (iii) the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.
... In addition, the cell-permeable inhibitor and facilitator peptides altered F 1 F 0 -ATPase activity and the PKC␦-dF 1 F 0 co-IP (Figs. [3][4][5] in opposing directions, whereas the scrambled sequence control peptide had no effect. ...
The F1F0-ATP synthase provides ∼90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions.
Previously, we demonstrated that protein kinase Cδ (PKCδ) inhibits F1F0 activity via an interaction with the “d” subunit of F1F0-ATP synthase (dF1F0) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831–29840). We have now identified a dF1F0-derived peptide (NH2-2AGRKLALKTIDWVSF16-COOH) that inhibits PKCδ binding to dF1F0 in overlay assays. We have also identified a second dF1F0-derived peptide (NH2-111RVREYEKQLEKIKNMI126-COOH) that facilitates PKCδ binding to dF1F0. Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting
sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCδ-dF1F0 interaction inhibitor, decreased 100 nm 4β-phorbol 12-myristate 13-acetate (4β-PMA)-induced co-immunoprecipitation of PKCδ with dF1F0 by 50 ± 15% and abolished the 30 nm 4β-PMA-induced inhibition of F1F0-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences,
was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCδ-dF1F0 facilitator peptide by itself induced the PKCδ-dF1F0 co-immunoprecipitation and inhibited F1F0-ATPase activity. In in vitro PKC add-back experiments, the PKCδ-F1F0 inhibitor blocked PKCδ-mediated inhibition of F1F0-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially
targeted modulators of the PKCδ-dF1F0 interaction in NCMs. These novel peptides will improve our understanding of cardiac F1F0 regulation and may have potential as therapeutics to attenuate cardiac injury.
... The interfaces between ␣and -subunits are the site of nucleotide binding and ATP synthesis. The F 1 and F 0 domains are connected by a central stalk consisting of the ␥, ␦, and ⑀ subunits and by a peripheral stalk that is made up of the OSCP, F6, b, and d subunits (5). The central stalk rotates along with a ring of 10 -14 "c" subunits during ATP synthesis, and the peripheral stalk acts as a stator to prevent the ␣and -subunits from rotating with the central stalk and c-subunits. ...
Mitochondrial protein kinase C isozymes have been reported to mediate both cardiac ischemic preconditioning and ischemia/reperfusion
injury. In addition, cardiac preconditioning improves the recovery of ATP levels after ischemia/reperfusion injury. We have,
therefore, evaluated protein kinase C modulation of the F1F0 ATPase in neonatal cardiac myocytes. Exposure of cells to 3 or 100 nm 4β-phorbol 12-myristate-13-acetate induced co-immunoprecipitation of δ protein kinase C (but not α, ϵ, or ζ protein kinase
C) with the d subunit of the F1F0 ATPase. This co-immunoprecipitation correlated with 40 ± 3% and 72 ± 9% inhibitions of oligomycin-sensitive F1F0 ATPase activity, respectively. We observed prominent expression of δ protein kinase C in cardiac myocyte mitochondria, which
was enhanced following a 4-h hypoxia exposure. In contrast, hypoxia decreased mitochondrial ζPKC levels by 85 ± 1%. Following
4 h of hypoxia, F1F0 ATPase activity was inhibited by 75 ± 9% and δ protein kinase C co-immunoprecipitated with the d subunit of F1F0 ATPase. In vitro incubation of protein kinase C with F1F0 ATPase enhanced F1F0 activity in the absence of protein kinase C activators and inhibited it in the presence of activators. Recombinant δ protein
kinase C also inhibited F1F0 ATPase activity. Protein kinase C overlay assays revealed δ protein kinase C binding to the d subunit of F1F0 ATPase, which was modulated by diacylglycerol, phosphatidylserine, and cardiolipin. Our results suggest a novel regulation
of the F1F0 ATPase by the δ protein kinase C isozyme.
... Proton flux down its electrochemical gradient through the F 0 segment produces a rotary torque in the shaft connecting the F 0 -F 1 segments to release ATP [34]. The conversion of ATP synthase to hydrolase in the mitochondria may be a mechanism to maintain membrane potential [35,36]. ...
The mitochondrial F1F0 ATP synthase is responsible for the majority of ATP production in mammals and does this through a rotary catalytic mechanism. Studies show that the F1F0 ATP synthase can switch to an ATP hydrolase, and this occurs under conditions seen during myocardial ischemia. This ATP hydrolysis causes wasting of ATP that does not produce work. The degree of ATP inefficiently hydrolyzed during ischemia may be as high as 50-90% of the total. A naturally occurring, reversible inhibitor (IF-1) of the hydrolase activity is in the mitochondria, and it has a pH optimum of 6.8. Based on studies with the nonselective (inhibit both synthase and hydrolase activity) inhibitors aurovertin B and oligomycin B reduce the rate of ATP depletion during ischemia, showing that IF-1 does not completely block hydrolase activity. Oligomycin and aurovertin cannot be used for treating myocardial ischemia as they will reduce ATP production in healthy tissue. We generated a focused structure-activity relationship, and several compounds were identified that selectively inhibited the F1F0 ATP hydrolase activity while having no effect on synthase function. One compound, BMS-199264 had no effect on F1F0 ATP synthase function in submitochondrial particles while inhibiting hydrolase function, unlike oligomycin that inhibits both. BMS-199264 selectively inhibited ATP decline during ischemia while not affecting ATP production in normoxic and reperfused hearts. BMS-191264 also reduced cardiac necrosis and enhanced the recovery of contractile function following reperfusion. These data also suggest that the reversal of the synthase and hydrolase activities is not merely a chemical reaction run in reverse.
... cke 2001 ) . Recently , convincing proof was given that a mechanical movement of pro - tein subunits , which form a rotor interacting with other parts of the F 0 F 1 - ATPase , are able to synthesize ATP by a " downhill " movement of protons through the F 0 unit or to pump protons " uphill " at the expense of ATP hydrolysis ( Senior et al . 2002 ; Gaballo et al . 2002 ) . In these cases , concepts of well - known macroscopic machines , like a mechanic pump or a water - driven mill , can be used , when scaled down , to describe for these highly specific machines dimensions of a few nanometers . ...
P-type ATPases are a large family of membrane proteins that perform active ion transport across biological membranes. In these proteins the energy-providing ATP hydrolysis is coupled to ion-transport that builds up or maintains the electrochemical potential gradients of one or two ion species across the membrane. P-type ATPases are found in virtually all eukaryotic cells and also in bacteria, and they are transporters of a broad variety of ions. So far, a crystal structure with atomic resolution is available only for one species, the SR Ca-ATPase. However, biochemical and biophysical studies provide an abundance of details on the function of this class of ion pumps. The aim of this review is to summarize the results of preferentially biophysical investigations of the three best-studied ion pumps, the Na,K-ATPase, the gastric H,K-ATPase, and the SR Ca-ATPase, and to compare functional properties to recent structural insights with the aim of contributing to the understanding of their structure-function relationship.
The dynamical systems found in Nature are rarely isolated. Instead they interact and influence each other. The coupling functions that connect them contain detailed information about the functional mechanisms underlying the interactions and prescribe the physical rule specifying how an interaction occurs. Here, we aim to present a coherent and comprehensive review encompassing the rapid progress made recently in the analysis, understanding and applications of coupling functions. The basic concepts and characteristics of coupling functions are presented through demonstrative examples of different domains, revealing the mechanisms and emphasizing their multivariate nature. The theory of coupling functions is discussed through gradually increasing complexity from strong and weak interactions to globally-coupled systems and networks. A variety of methods that have been developed for the detection and reconstruction of coupling functions from measured data is described. These methods are based on different statistical techniques for dynamical inference. Stemming from physics, such methods are being applied in diverse areas of science and technology, including chemistry, biology, physiology, neuroscience, social sciences, mechanics and secure communications. This breadth of application illustrates the universality of coupling functions for studying the interaction mechanisms of coupled dynamical systems.
The identification of proteins involved in brain ischemia might allow the discovery of putative biomarkers or therapeutic targets for ischemic stroke. Our aim is to study the distribution of proteins within mouse brain after an ischemic insult using MALDI imaging-mass-spectrometry and to identify relevant proteins involved in brain damage. We occluded the middle cerebral artery of C57BL/6J mice. Brain slices were analyzed by MALDI-TOF and infarct (IC) and contralateral (CL) regions were compared using ClinProTools. The ion distribution maps of relevant m/z values were obtained by FlexImagin3.0. Protein identification was conducted through a bottom-up approach consisting on complementary sample fractionation methods. Some identifications were confirmed by immunohistochemistry and western blot. We identified 102 m/z values with different abundances between IC and CL (p<0.05), from which 21 m/z peaks were selected as more relevant. Thirteen of them were found increased in the infarct region and 4 m/z values showed AUC>90% between IC and CL. Identification analyses confirmed altered expressions of ATP5i, COX6C and UMP-CMP kinase in IC compared to CL.
Biological significance:
Using MALDI-IMS we identified for the first time new proteins that might be involved in brain ischemia representing potential diagnostic biomarkers or target molecules for neurological recovery.
